Solid tumors are difficult to treat, especially when they reach a metastatic stage. Standard therapies often show severe side-effects. Therefore, the evaluation of new and efficient treatment options are in urgent need. With targeted therapies using immunotoxins (IT) or in best case human cytolytic fusion proteins (hCFP), tumor cells could be killed specifically without affecting healthy cells. Hence, this work was focused on the evaluation of a Granzyme B (GrB)-based hCFP, which could induce apoptotic signal-cascades in tumor cells. GrB was fused to a cell-binding antibody-fragment, a new human epidermal growth factor receptor (EGFR)-specific single-chain Fragment variable (scFv), to demonstrate binding and subsequent internalization by receptor bearing cells. The EGFR is expressed on a variety of solid tumors and is associated with poor prognosis. Rhabdomyosarcoma (RMS), pancreatic-, prostate-, and epoidermoid carcinoma, as well as the breast cancer type triple negative breast cancer (TNBC) are difficult to treat and were the main focus of this study. To initially analyze the functionality of the human and a second EGFR-specific scFv, both were fused to the SNAP-Tag and characterized. ScFv1711-SNAP was derived from Panitumumab and scFv2112-SNAP from Cetuximab, which are established EGFR-specific monoclonal antibodies (mAb) in the clinic. Both scFv-SNAP fusion proteins showed specific binding and internalization into the target cells within minutes. A critical factor concerning ITs and hCFPs is the affinity constant of the used scFvs. In order to use SPR for determining the affinity of the scFv-SNAP fusion proteins, a regenerative system was established. For this purpose, the fast and easy coupling reaction of BG-Biotin to the scFv-SNAP fusion was combined with the commercial available “Biotin CAPture kit”, which works on the basis of Streptavidin/Biotin binding. Both scFvs show KD-values of ~ 4 nM. Additionally the competitive behavior of the scFvs was tested and compared to the parental mAbs, respectively. To get a first hint of the in vivo-potential, scFv2112-SNAP was successfully tested as NIR-probe in a subcutaneous prostate cancer-mouse model. After the functional characterization of the new scFvs, both were fused to a truncated version of Pseudomonas Exotoxin A (ETA') as proof of concept. Using cell lines from different tumor entities and with varying EGFR-expression levels, specific binding, internalization within 30-60 min, as well as IC50-values in the picomolar range (4-460 pM depending on the cell line) were determined. The cell line with the highest EGFR-expression level was most efficiently eliminated in cell viability assays. Both ITs also induced statistically significant apoptosis. In addition, specific binding on human tumor biopsies could be confirmed. The aim of the study was to develop and evaluate an hCFP for the treatment of solid tumors. This was demonstrated by using GbR201K-scFv1711 with a Serpin B9-resistent mutated GrB-variant. After expression and purification with yields in the range of 17-30 mg/l protein, specific binding and internalization was confirmed on selected cell lines, by using enzymatically activated GbR201K-scFv1711. GbR201K-scFv1711 demonstrated a high serum stability, even after 72h. Significant apoptotic and anti-proliferative effects (IC50 = 133.3 nM) were reached with A431-cells (epidermoid carcinoma). By using the endosomolytic substance Chloroquine (CQ) induction of apoptosis and an IC50-value of 21 nM could be determined with RD-cells (RMS) as well. The IC50-value of A431-cells improved to 45 nM. Furthermore, specific binding on human RMS-biopsies was demonstrated. These data confirm that especially this novel hCFP is an interesting candidate for further pre-clinical studies